Before entering the canonical gluconeogenic pathway, the vast majority of gluconeogenic carbon flux is routed through the TCA cycle within the mitochondrial matrix. Under normal conditions, mitochondrial pyruvate is the key metabolic intermediate supporting gluconeogenesis. To reach the matrix and drive gluconeogenesis, pyruvate requires a specific carrier, the Mitochondrial Pyruvate Carrier (MPC). Thus, the MPC occupies a central node linking cytosolic with mitochondrial metabolism and gluconeogenesis. The mechanisms regulating the function of the MPC in the liver have remained unexplained. The recent identification of the MPC molecule now enables the investigation of these mechanisms. The overall goal of this proposal is to understand the relationship between MPC function in the liver and the pathologically elevated gluconeogenesis during T2D. This goal will be addressed by pursuing two specific aims: 1) Determine the mechanisms that regulate liver MPC function in response to fasting and refeeding and in T2D states; and 2) Determine the therapeutic effect of decreasing Liver MPC activity on hyperglycemia during T2D states. The studies in aim 1 will test the hypothesis that the MPC is transcriptionally and post-translationall regulated by hormonal mechanisms during fasting and refeeding that increase and decrease MPC activity, respectively, and that misregulation of the MPC in T2D states increases its activity thereby increasing mitochondrial pyruvate uptake and gluconeogenesis. The studies in aim 2 will test the hypothesis that decreasing liver MPC activity in vivo during T2D states will decrease gluconeogenesis and ameliorate hyperglycemia. They will also define how liver-specific MPC knockout alters gluconeogenic and TCA cycle carbon flux using metabolic tracers in normal and T2D mice and primary hepatocytes. This research is significant because successful completion will provide fundamental information on the role of the MPC in driving elevated hepatic gluconeogenesis and resultant hyperglycemia in T2D. This research is innovative because completion will generate novel, and now only recently obtainable, knowledge on the regulation and function of the MPC molecule as a critical node linking cytosolic and mitochondrial metabolism with gluconeogenesis. We expect our studies will unveil a new paradigm for explaining and potentially decreasing the excessive gluconeogenesis that characterizes T2D.